Abstract Breast cancer remains a serious global disease associated with high rates of mortality. According to The Centers for Disease Control, 224,147 women and 2,125 men in the U.S. were diagnosed with breast cancer in 2012. Over the next ten years, the CDC estimates that 19.7 million individuals will suffer from breast cancer. Current treatments for the majority of breast cancers often depend upon interfering with hormone (ER, PR) or growth factor (HER2) receptors on those cells. Individuals who receive receptor-targeted treatments have a good prognosis for long term survival. Triple-negative breast cancer (TNBC) is a subtype of cancer caused by proliferation of breast cells that are deficient in expression of those three receptors, thus abrogating the option of targeting of those receptors as a treatment strategy. In contrast to other types of breast cancers, individuals who are diagnosed with TNBC have exceptionally poor treatment options, a higher frequency of metastatic disease, and shorter survival times. Thus, new molecules and pathways in TNBC cells that can be efficiently targeted to treat individuals diagnosed with the disease are urgently needed. In addition to their lower efficacies, current chemotherapeutic agents for TNBC elicit a number of serious side effects that are engendered by off-target activities of the treatment agents. These side effects include overwhelming fatigue, infertility, nausea, and cardiac toxicity. Thus, it is critical that more specific targets in TNBC cells are identified for therapeutic intervention. Recently, we demonstrated that LT-IIc, a bacterial type II ADP-ribosylating toxin (AR-Tx), is lethal for TNBC cells, but has no lasting effects on non- transformed breast epithelial cells. Preliminary observations suggest that the lethal effects of LT-IIc for TNBC cells is due to induction of an irreversible autophagy that is absent in normal breast epithelial cells. We will employ LT-IIc as a PROBE to identify cytotoxic mechanisms that are specifically induced in TNBC cells. A combination of pharmacological, genetic, and fluorescent approaches will be employed to evaluate the contributions of ganglioside-engagement, ADP-ribosylation, and intracellular cAMP on TNBC-specific cytotoxicity. These experiments will be facilitated by the availability of a set of recombinant AR-Tx chimeras that ?mix-and-match? the ADP-ribosylating and ganglioside-binding subunits of TNBC-cytotoxic LT-IIc and non-cytotoxic LT-IIb. The roles of preferential ganglioside expression and engagement in TNBC-specific cytotoxic responses, a potentially productive and unexplored area of research, will be evaluated using novel TLC immunoblotting and mass-spectroscopy. The effects of LT-IIc on tumor growth and metastasis will be interrogated using a mouse TNBC tumor model, in vivo imaging, and in vitro culture methods. Our immediate goal is to employ LT-IIc as a molecular and cellular PROBE to uncover new and exploitable TNBC-specific cytotoxic pathways. Our LONG TERM goal is to identify pharmacological agents that target those pathways and to develop new and safer approaches for treatment of TNBC.